Chromatin structure is increasingly recognized as a key component in gene regulation. Chromatin structure is highly dynamic, and is determined by chromatin remodeling enzymes. There are three classes of remodeling enzymes; ATP-dependent remodeling enzymes use the energy of ATP hydrolysis to alter histone-DNA contacts, histone modifying enzymes covalently modify histone proteins, while DNA modifying enzymes add and remove methyl marks from the DNA itself. The importance of remodeling is especially appreciated in the immune system, where there are many examples of changes in chromatin structure associated with changes in gene expression. Moreover, it is possible to obtain defined populations of lymphocytes for experimentation. However, little is known about the enzymes mediating these changes. We use cell-based and cell free approaches to address this deficit by examining the role of remodeling in gene regulation, and the mechanism of remodeling.[unreadable] [unreadable] [unreadable] Cell-based studies [unreadable] [unreadable] We are investigating the role of ATP-dependent remodeling enzymes in T cell gene regulation using cultured cells. We developed conditions to transduce T cell lines and T cell lines with high efficiency (greater than 60%), and to enrich for transduced cells. We used this methodology to knock down expression of remodeling enzyme subunits. This allowed us to ask what genes required remodeling enzyme expression for proper regulation. We have identified cytokine genes that are directly regulated by remodeling enzymes.[unreadable] [unreadable] Cell-free studies[unreadable] [unreadable] We are investigating the mechanism of ATP-dependent remodeling enzymes using a cell-free biochemical system. We assemble chromatin with physiologic properties using either purified proteins or extracts from fly embryos. We then ask how chromatin structure changes occur, and what effect they have on chromatin function, using transcription or recombination as a readout. The current project uses an enhancer fragment from the immunoglobulin heavy-chain locus, and was initiated as a collaboration with Dr. Ranjan Sen while he was at Brandeis and I was at MGH/Harvard, and is related to work on the Ets family of transcription factors (Lu et al., 2004). Unexpectedly, we found that a transcription factor could prevent chromatin remodeling in an extract (Ishii et al., 2004). We have extended these findings using a purified system. This work has revealed that one transcription factor can reverse the remodeling directed by a first, and two different ATP-dependent remodeling enzymes mediate opposing effects.